Nuclear pore complexes (NPCs) form the only sites for entry and exit from the nucleus. This includes the diffusion of small molecules and the selective, active transport of large proteins and RNA. Proper NPC biogenesis is critical for cell division, differentiation, and responses to changes in metabolic activities. Understanding NPC assembly at the molecular level will be key for designing strategies to inhibit cell growth and gene expression, for example in oncogenesis. However, the molecular pathway for de novo insertion of NPCs into the intact nuclear envelope (NE) is largely undefined. The most outstanding question in the field involves delineating how nuclear pore formation is executed and regulated. The pathway for coordinating interactions between at least 30 different NPC proteins (Nups) and multiple pore membrane proteins (Poms) is unknown. This project aims to reveal the molecular biogenesis mechanism for the NPC transport apparatus. Our specific aims will each utilize aggressive yeast S. cerevisiae genetics/biochemistry/cell biology-based approaches directly coupled with state-of-the-art microscopy and biochemistry in metazoan cells and the Xenopus egg extract in vitro assembly system. This innovative merger of strengths in multiple model systems is only possible through the full-fledged collaborative efforts of the two PIs. We speculate that de novo assembly is a step-wise process. Our first specific aim will address early assembly events at the NE and chromatin. Using a novel set of yeast NPC assembly mutants and in vitro Xenopus assays, we will identify the essential Nups and assembly factors that are targeted to chromatin, outer and/or inner nuclear membranes. The targeting mechanism and role in assembly will be directly tested. The second aim will focus on the mechanism for fusion of the outer and inner nuclear membranes, i.e. formation of a pore across the NE, and builds on our recent discovery of ER/NE proteins required for NPC assembly and our novel pore fusion assay in Xenopus extracts. We hypothesize that the highly curved pore membranes are formed by the action of Poms, stabilized by transient association of the reticulons, and maintained by the recruitment of a membrane coat formed by the Nup107-160/Nup84 complex. In the third aim, we will analyze the coordination between sequential steps in the biogenesis process. Assembly intermediates will be examined by scanning electron microscopy and purified from yeast mutant cells arrested at distinct steps. We will also pinpoint the order of metazoan Nup recruitment after membrane hole formation by fluorescence imaging of NPC assembly with single pore resolution in real time. Together, these studies will define the membrane fusion machinery and the sequence of self-assembly steps for NPC biogenesis in intact NEs. PUBLIC HEALTH RELEVANCE: Transport of proteins and RNA between the nucleus and cytoplasm is essential for all aspects of normal cell function, and requires large protein machines (nuclear pore complexes) to allow cargo movement. Trafficking is perturbed in some disease states, such as cancer and viral infections, and there are also genetically heritable diseases that are linked to changes in the genes encoding transport factors and nuclear pore complexes. Knowledge of how nuclear pore complexes are assembled and function will be important for finding therapeutic strategies to regulate nuclear transport in disease states and moderate viral proliferation/pathogenesis.